Tumor necrosis factor inhibitors

ABSTRACT

Peptides which consist of 4-25 amino acids and which bind to tumor necrosis factor-alpha, prevent tumor necrosis factor-alpha from binding to its receptors and inhibit tumor necrosis factor-alpha activity are disclosed. Methods of inhibiting tumor necrosis factor-alpha activity and of treating individuals suffering from tumor necrosis factor-alpha-mediated diseases and disorders are disclosed.

FIELD OF THE INVENTION

The present invention relates to compounds that inhibit tumor necrosisfactor-alpha (TNFα) activity by binding to TNF-α and thereby preventingthe TNFα from binding to TNF receptors. Thus, TNFα activity which ismediated through its binding to its receptor on cells is inhibited.

BACKGROUND OF THE INVENTION

The cytokine known as tumor necrosis factor-α (TNFα; also termedcachectin) is a protein secreted primarily by monocytes and macrophagesas a soluble homotrimer of 17 kD protein subunits in response toendotoxin or other stimuli (Smith, R. A. et al., J. Biol. Chem. 1987,262, 6951-6954). A membrane-bound 26 kD precursor form of TNFα has alsobeen described (Kriegler, M. et al., Cell 1988, 53, 45-53). TNFα wasoriginally discovered in the serum of animals injected sequentially witha bacterial vaccine (bacillus Calmette-Guerin, BCG) and endotoxin(Carswell, E. A. et al., Proc. Natl. Acad. Sci. USA 1975, 72, 3666).

The expression of the gene encoding TNFα is not limited to cells of themonocyte/macrophage family. Several human non-monocytic tumor cell lineswere shown to produce TNFα. TNFα is also produced by CD4⁺ and CD8⁺peripheral blood T lymphocytes, and by various cultured T and B celllines.

TNFα plays an integral role in destroying tumors, mediating responses totissue injury, and protecting hosts from infections by variousmicroorganisms (Goeddel et al., Cold Spring Harbor Symp. Quant. Biol.1986, 51, 597-609; Beutler et al., Ann. Rev. Immunol. 1989, 7, 625-655;Malik et al. in Tumor Necrosis Factor: Structure, Function and Mechanismof Action, Aggarwal and Vilcek, Eds. (Marcel Dekker, Inc., 1992); Fiers,FEBS Letters 1991, 285, 199-212; and Buetler et al., Buetler, B., Ed. inTumor Necrosis Factors: the Molecules and Their Emerging Role inMedicine (Raven Press, New York, NY, 1992)). However, its activityappears to be excessive in some disease states and inflammatoryreactions such as rheumatoid arthritis, cachexia, and septic shock(Pujol-Borrell et al., Nature 1987 326, 304-306; Oliff, Cell 1988 54,141-142; Tracey et al., Nature 1987, 330, 662-664). The excess TNFαresults in an exaggerated immune response exemplified by overstimulationof interleukin-6 and granulocyte/macrophage-colony stimulating factor(GM-CSF) secretion, enhanced cytotoxicity of polymorphonuclearneutrophils, and prolonged expression of cellular adhesion molecules,all of which can have detrimental effects. The benefits of inhibitingTNFα activity during inflammatory reactions in animal models have beendemonstrated using neutralizing monoclonal antibodies to TNFα (Tracey etal., Nature 1987, 330, 662-664; Silva et al., J. Infect. Sis. 1990,162,421-427; and Williams et al., Proc. Natl. Acad. Sci. 1992, 89,9784-9788).

The mechanism of action of TNFα is derived from accumulating evidencewhich indicates that TNFα is a regulatory cytokine with pleiotropicbiological activities. These activities include: inhibition oflipoprotein lipase synthesis ("cachectin"), activation ofpolymorphonuclear leukocytes, inhibition of cell growth or stimulationof cell growth, cytotoxic action on certain transformed cell types,antiviral activity, stimulation of bone resorption, stimulation ofcollagenase and prostaglandin E2 production, and immunoregulatoryactions, including activation of T cells, B cells, monocytes,thymocytes, and stimulation of the cell-surface surface expression ofmajor histocompatibility complex class I and class II molecules.

TNFα is noted for its pro-inflammatory actions which result in tissueinjury, such as induction of procoagulant activity on vascularendothelial cells (Pober, J. S. et al., J. Immunol. 1986, 336, 1680),increased adherence of neutrophils and lymphocytes (Pober, J. S. et al.,J. Immunol. 1987, 138, 3319), and stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells (Camussi, G. et al., J. Exp. Med. 1987, 166, 1390).

Recent evidence implicates TNFα in the pathogenesis of many infections(Cerami, A. et al., Immunol. Today 1988, 9, 28), immune disorders,neoplastic pathology, e.g., in cachexia accompanying some malignancies(Oliff, A. et al., Cell 1987, 50, 555), and in autoimmune pathologiesand graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med.1987, 166, 1280). The association of TNFα with cancer and infectiouspathologies is often related to the host's catabolic state. A majorproblem in cancer patients is weight loss, usually associated withanorexia. The extensive wasting which results is known as "cachexia"(Kern, K. A. al., J. Parent. Enter. Nutr. 1988, 12, 286-298). Cachexiaincludes progressive weight loss, anorexia, and persistent erosion ofbody mass in response to a malignant growth. The fundamentalphysiological derangement may be related to a decline in food intakerelative to energy expenditure. The cachectic state is thus associatedwith significant morbidity and is responsible for the majority of cancermortality. A number of studies have suggested that TNFα is an importantmediator of the cachexia in cancer, infectious pathology, and in othercatabolic states.

TNFα is thought to play a central role in the pathophysiologicalconsequences of Gram-negative sepsis and endotoxic shock (Michie, H. R.et al., Br. J. Surg. 1989, 76, 670-671; Debets, J. M. H. et al., SecondVienna Shock Forum, 1989, p.463-466; Simpson, S. Q. et al., Crit. CareClin. 1989, 5, 27-47), including fever, malaise, anorexia, and cachexia.Endotoxin is a potent monocyte/macrophage activator which stimulatesproduction and secretion of TNFα (Kornbluth, S. K. et al., J. Immunol.1986, 137, 2585-2591) and other cytokines. Because TNFα could mimic manybiological effects of endotoxin, it was concluded to be a centralmediator responsible for the clinical manifestations ofendotoxin-related illness. TNFα and other monocyte-derived cytokinesmediate the metabolic and neurohormonal responses to endotoxin (Michie,H. R. et al., N. Eng. J. Med. 1988, 318, 1481-1486). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, A. et al., Arch. Surg. 1988, 123,162-170). Elevated levels of circulating TNFα have also been found inpatients suffering from Gram-negative sepsis (Waage, A. et al., Lancet1987, 1, 355-357). Treatment of cancer patients with TNFα (because ofits tumoricidal action) revealed that doses greater than 545 μg/m² /24hours caused alterations similar to those induced by injection ofendotoxin (4 ng/kg) into healthy humans (Michie, H. R. et al., Surgery1988, 104, 280-286), supporting TNFα's role as the principal hostmediator of septic and endotoxemic responses. Chronic intravenous TNFαinfusion into humans or rats was associated with anorexia, fluidretention, acute phase responses, and negative nitrogen balance (i.e.,classic catabolic effects), leading to the conclusion that TNFα may beresponsible for many of the changes noted during critical illness(Michie, H. R. et al., Ann. Surg. 1989, 209, 19-24).

The numerous biological effects of TNFα and the closely relatedcytokine, TNFβ (lymphotoxin), are mediated by two transmembranereceptors, both of which have been cloned. The p55 receptor (also termedTNF-R55, TNF-RI, or TNFRβ) is a 55 kd glycoprotein shown to transducesignals resulting in cytotoxic, anti-viral, and proliferative activitiesof TNFα.

The p75 receptor (also termed TNF-R75, TNF-RII, or TNFRα) is a 75 kdglycoprotein that has also been shown to transduce cytotoxic andproliferative signals as well as signals resulting in the secretion ofGM-CSF. The extracellular domains of the two receptors are 28% identicalin primary structure and have in common a set of four subdomains definedby numerous conserved cysteine residues. The p75 receptor differs,however, by having a region adjacent to the transmembrane domain that isrich in proline residues and contains sites for 0-linked glycosylation.Interestingly, the cytoplasmic domains of the two receptors share noapparent homology which is consistent with observations that they cantransduce different signals to the interior of the cell.

TNFα inhibitors have been detected in normal human urine and in serum ofpatients with cancer or endotoxemia. These have since been shown to bethe receptor extra-cellular domains derived by proteolytic cleavage ofthe transmembrane forms. Many of the same stimuli that result in TNFαrelease also result in the release of the soluble receptors, suggestingthat these soluble TNFα inhibitors may serve as part of a negativefeedback mechanism to control TNFα activity (Porteu, F. and C. Nathan(1990) J. Exp. Med. 172:599-607; and, Adreke, D. et al., (1992) J. Exp.Med. 175:323-329).

There is a need for compounds which effectively inhibit TNFα activity.There is a need to provide compounds that bind to TNFα with highaffinity and can prevent TNFα from binding to its receptors. There is aneed for compounds which can neutralize TNFα activity in vivo.

SUMMARY OF THE INVENTION

The present invention relates to peptides which comprise an amino acidsequence consisting of 4 to 25 amino acids and which inhibit TNFαactivity. The peptides of the invention comprises at least a four aminoacid residue fragment of one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4 or SEQ ID NO:5.

The present invention relates to a method of inhibiting tumor necrosisfactor-alpha activity comprising the step of contacting tumor necrosisfactor-alpha with a peptide that comprises an amino acid sequence whichconsists of 4 to 25 amino acids and which inhibits TNFα activity. Thepeptide used in the method of inhibiting tumor necrosis factor-alphaactivity comprises at least a four amino acid residue fragment of one ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

The present invention relates to a method of treating an animalsuspected of suffering from a disease or disorder mediated by tumornecrosis factor-alpha activity comprising the step of administering tosaid individual a therapeutically effective amount of a peptide thatinhibits tumor necrosis factor- alpha. The peptide comprises an aminoacid sequence which consists of 4 to 25 amino acids and comprises atleast a four amino acid residue fragment of one of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a graph which shows data generated from experiments usingpeptides which are embodiments of the invention. The experiments wereperformed to evaluate dose dependent inhibition of TNFα by the peptidestested.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, compounds are provided which bind tothe TNFα and thereby prevent it from binding to p55 and p75 receptors.By inhibiting such TNFα/TNF receptor binding, the compounds of theinvention inhibit the biological activity of TNFα. By blocking TNFα frombinding to its receptors, the compounds of the invention prevent TNFαfrom producing the biological effect associated with the TNFα-TNFreceptor binding.

Compounds according to some aspects of the invention are peptidesconsisting of 4 to 25 amino acids. In some embodiments, the peptidesconsist of 20 amino acids or less. In some embodiments, the peptidesconsist of at least 8 amino acids. In some embodiments, the peptidesconsist of 8-20 amino acids. In some embodiments, the peptides consistof 10-15 amino acids.

According to some embodiments of the present invention, peptidescomprise of amino acid sequences selected from the group consisting of:fragments of SEQ ID NO:1 that have at least four amino acid residues,fragments of SEQ ID NO:2 that have at least four amino acid residues,fragments of SEQ ID NO:3 that have at least four amino acid residues,fragments of 5 SEQ ID NO:4 that have at least four amino acid residues,and fragments of SEQ ID NO:5 that have at least four amino acidresidues.

In such embodiments, the peptides may further comprise additional aminoacid residues.

According to some embodiments of the present invention, peptides consistof amino acid sequences selected from the group consisting of: fragmentsof SEQ ID NO:1 that have at least four amino acid residues, fragments ofSEQ ID NO:2 that have at least four amino acid residues, fragments ofSEQ ID NO:3 that have at least four amino acid residues, fragments ofSEQ ID NO:4 that have at least four amino acid residues and fragments ofSEQ ID NO:5 that have at least four amino acid residues.

According to some embodiments of the present invention, peptidescomprise amino acid sequences selected from the group consisting of: SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

According to some embodiments of the present invention, peptides consistof amino acid sequences selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

In some embodiments, the peptides are conformationally restricted suchas those which are cyclicized, circularized or otherwise restricted bypeptide and/or non-peptide bonds to limit conformational variationand/or to increase stability and/or half-life of the peptides. In someembodiments, peptides are provided as linear peptides.

In some embodiments, peptides of the present invention comprise one ormore D amino acids. As used herein, the term "D amino acid peptides" ismeant to refer to peptides according to the present invention whichcomprise at least one and preferably a plurality of D amino acids. Damino acid peptides consist of 4-25 amino acids. D amino acid peptidesretain the biological activity of the peptides of the invention thatconsist of L amino acids, i.e. D amino acid peptides inhibit TNFα. Insome embodiments, the use of D amino acid peptides is desirable as theyare less vulnerable to degradation and therefore have a longer halflife. D amino acid peptides comprising mostly all D amino acids or Damino acid peptides that consist of only D amino acids may compriseamino acid sequences in the reverse order of amino acid sequences ofpeptides selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, fragments of SEQ ID NO:1 thathave at least four amino acid residues, fragments of SEQ ID NO:2 thathave at least four amino acid residues, fragments of SEQ ID NO:3 thathave at least four amino acid residues, fragments of SEQ ID NO:4 thathave at least four amino acid residues and fragments of SEQ ID NO:5 thathave at least four amino acid residues.

As used herein, the term "derivatives" refers to peptides of theinvention which have the amino terminal and/or the carboxy terminalblocked, particularly those in which the amino group of the N terminalresidue is acetylated and/or the carboxy group of the C terminal residueis amidated. Peptides that comprise an amino acid sequence whichconsists of 4 to 25 amino acids and which comprise at least a four aminoacid residue fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5 include derivatives.

In some preferred embodiments, the peptides of the invention areselected from the group consisting of: SEQ ID NO:1 in which the carboxyterminal phenylalanine is phenylalanine amide; SEQ ID NO:2 in which thecarboxy terminal tyrosine is tyrosine amide; SEQ ID NO:3 in which thecarboxy terminal alanine is alanine amide; SEQ ID NO:4 in which thecarboxy terminal glutamine is glutamine amide; and SEQ ID NO:5 in whichthe carboxy terminal glutamine is glutamine amide.

Contemplated equivalents include conservative analogs and mimetics. Asused herein, the term "conservative analog" is also meant to refer to apeptide consisting of 4-25 amino acids which inhibits TNFα and whichcomprises an amino acid sequence that includes at least a four aminoacid sequence that is substantially identical to at least a four aminoacid fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 orSEQ ID NO:5. As used herein, the term "substantially identical" refersto an amino acid sequence that is the same as the amino acid sequence ofat least a four amino acid fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or SEQ ID NO:5, except some of the residues aresubstituted with conservative amino acids. Conservative substitutions ofamino acids are determined, for example,following what are referred toas Dayhof's rules for amino acid substitution (Dayhof, M.D. (1978) Nat.Biomed. Res. Found., Washington, D.C. Vol. 5, supp. 3). Equivalentresidues are listed and amino acid residues in a peptide sequence may besubstituted with comparable amino acid residues. Such substitutions arewell known and are based the upon charge and structural characteristicsof each amino acid. Those having ordinary skill in the art can readilydesign and produce conservative analogs. In addition, the term"conservative analogs" is meant to encompass peptides which have aminoacid that comprise at least a four amino acid fragment of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, in which an aminoacid residue from the fragment is deleted and/or an amino acid isinserted within the fragment sequence. Conservative analogs inhibit TNFαin the same manner as the peptides of the invention. By interacting withTNFα in such a way and thereby inhibiting TNFα activity, conservativeanalogs perform essentially the same function by essentially the samemeans to achieve essentially the same result as the peptides of theinvention.

In addition to conservative analogs, the present invention contemplatescompounds which display substantially the same surface as the peptidesof the invention. As used herein, the term "mimetics" is meant to referto compounds that are not peptides but that comprise a similar surfaceas the peptides of the invention and can thus interact with the TNFreceptor in a similar fashion as the peptides of the invention. Mimeticsinhibit TNFα by interacting with TNFα in the same manner as the peptidesof the invention. Mimetics have a molecular surface similar to one of apeptide comprising at least a four amino acid fragment of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 and that molecularsurface interacts with TNFα. By providing a similar surface involved inintermolecular interactions, mimetics perform essentially the samefunction by essentially the same means to achieve essentially the sameresult as the peptides of the invention.

Peptides of the invention, including D amino acid peptides, may beprepared using the solid-phase synthetic technique initially describedby Merrifield, in J. Am. Chem. Soc., 15:2149-2154 (1963). Other peptidesynthesis techniques may be found, for example, in M. Bodanszky et al.,(1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent andClark-Lewis in Synthetic Peptides in Biology and Medicine, p. 295-358,eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985); as wellas other reference works known to those skilled in the art. A summary ofpeptide synthesis techniques may be found in J. Stuart and J. D. Young,Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, Ill.(1984), which is incorporated herein by reference. The synthesis ofpeptides by solution methods may also be used, as described in TheProteins, Vol. II, 3d Ed., p. 105-237, Neurath, H. et al., Eds.,Academic Press, New York, N.Y. (1976). Appropriate protective groups foruse in such syntheses will be found in the above texts, as well as in J.F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, NewYork, N.Y. (1973), which is incorporated herein by reference. Ingeneral, these synthetic methods involve the sequential addition of oneor more amino acid residues or suitable protected amino acid residues toa growing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectivelyremovable protecting group. A different, selectively removableprotecting group is utilized for amino acids containing a reactive sidegroup, such as lysine.

Block synthesis techniques may also be applied to both the solid phaseand solution methods of peptide synthesis. Rather than sequentialaddition of single amino acid residues, preformed blocks comprising twoor more amino acid residues in sequence are used as either startingsubunits or subsequently added units rather than single amino acidresidues.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

In order to determine whether a peptide inhibits TNFα, one or more ofseveral assays may be performed. Included among these are assays whichmeasure the ability a TNFα inhibitor candidate, i.e. a test compound, toinhibit TNFα from binding to a fusion protein that is composed of a TNFreceptor or a TNFα-binding portion thereof, fused to an immunoglobulinmolecule or a portion thereof. In other assays, the ability a testcompound to inhibit TNFα from binding to an isolated TNF receptor ismeasured. Other assays include those which the ability of a TNFαinhibitor candidate, i.e. a test compound, to inhibit TNFα activity whenTNFα is contacted with cells that react to the presence of TNFα. Forexample, TNFα is cytotoxic to some cells, such as WEHI cells, and assayscan be used to measure the ability a test compound, to inhibit TNFαcytotoxicity.

There are numerous other assays which can be used to determine a testcompound's ability to inhibit TNFα. In some assays, specific non-lethaleffects of TNFα on some cells is used as an end point to evaluate theTNFα inhibitory activity of a test compound. Known effects of TNFα onfibroblast cells include effects on mitogenesis, IL-6 secretion and HLAclass II antigen induction. Comparisons can be made between TNFα'seffect on fibroblasts in the presence or absence of a test compoundusing these detectable phenotypic changes as endpoints. Similarly, knowneffects of TNFα on monocyte cells include effects on secretion ofcytokines such as GMCSF, IL-6 and IL-8. Comparisons can be made betweenTNFα's effect on cytokine secretion by monocytes in the presence orabsence of a test compound. Additionally, TNFα is known to have effectson secretion of cytokine by endothelial cells and similar assays may bedesigned and performed. Further, TNFα is also known to effect adhesionmolecule induction, ICAM-1, E-selectin, VCAM and tissue factorproduction in endothelial cells. Comparisons can be made between TNFα' seffect on endothelial cells in the presence or absence of a testcompound using these detectable phenotypic changes as endpoints as well.Likewise, TNFα is known to effect neutrophils in specific ways.Comparisons can be made between TNFα's effect on neutrophils in thepresence or absence of a test compound using activation, priming,degranulation and superoxide production as detectable endpoints forevaluation of TNFα inhibitory activity. These and other assays are wellknown to those having ordinary skill in the art. Such assays may bedesigned and performed routinely form readily available startingmaterials.

The TNFα inhibitors according to the invention are useful for treating avertebrate having a pathology or condition associated with levels of asubstance reactive with a TNF receptor, in particular TNFα, in excess ofthe levels present in a normal healthy subject. Such pathologiesinclude, but are not limited to: sepsis syndrome, including cachexia;circulatory collapse and shock resulting from acute or chronic bacterialinfection; acute and chronic parasitic or infectious processes,including bacterial, viral and fungal infections; acute and chronicimmune and autoimmune pathologies, such as systemic lupus erythematosusand rheumatoid arthritis; alcohol-induced hepatitis; chronicinflammatory pathologies such as sarcoidosis and Crohn's pathology;vascular inflammatory pathologies such as disseminated intravascularcoagulation; graft-versus-host pathology; Rawasaki's pathology; andmalignant pathologies involving TNFα-secreting tumors.

Such treatment comprises administering a single or multiple doses of thecompounds of the invention. Preferred for human pharmaceutical use arepharmaceutical compositions that comprise the compounds of the presentinvention in combination with a pharmaceutically acceptable carrier ordiluent.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. In the case of thepeptides of the invention, the primary focus is the ability to reach andbind with TNFα. Because proteins are subject to being digested whenadministered orally, parenteral administration, i.e., intravenous,subcutaneous, intramuscular, would ordinarily be used to optimizeabsorption. In some preferred embodiments, pharmaceutical compositionswhich comprise the compounds of the present invention are administeredintravenously or subcutaneously.

Pharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily dosage of active ingredient can be about 0,001 to 1 grams perkilogram of body weight, in some embodiments about 0.1 to 100 milligramsper kilogram of body weight. Ordinarily dosages are in the range of 0.5to 50 milligrams per kilogram of body weight, and preferably 1 to 10milligrams per kilogram per day. In some embodiments, the pharmaceuticalcompositions are given in divided doses 1 to 6 times a day or insustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95 by weight based on the total weight of the composition.

For parenteral administration, the TNFα inhibitor can be formulated as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils may also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

For example, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

EXAMPLES Example 1

p55 receptor/IgG fusion protein binding assay

In order to screen compounds for their ability to block TNFα binding tothe TNF p55 receptor, an assay has been designed using TNFα and ap55/IgG fusion protein in place of monovalent, non-fusion p55 TNFreceptor protein. This assay was designed to identify peptides whichbind to human TNFα and thereby prevent the capture of the TNFα by amicrotiter plate coated with p55-Ig fusion protein. A constantconcentration of human TNFα is preincubated with the test peptide andthen incubated on the p55-Ig coated microtiter wells. Bound TNFα isdetected using a specific antisera and an alkalinephosphatase-conjugated probe. An active peptide will reduce the amountof human TNFα bound to the well relative to control wells in which TNFαbut no peptide was added.

A 96-well, U-bottom polyvinylchloride microtiter plate was coated with50 μl/well of p55-Ig fusion protein at 5 μg/ml in 0.01M sodiumphosphate, 0.15M sodium chloride (PBS) by incubation overnight at 4° C.or 2 hours at 37° C. The fusion protein, which consists of a p55 TNFreceptor protein portion and an IgG portion, can be produced asdisclosed in U.S. application Ser. No.08/010,406 filed Jan. 29, 1993which is incorporated herein by reference. The plate was washed threetimes with 0.05% Tween-20 in PBS, then blocked, by adding 150 μl/well ofassay buffer (10 mM N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid(HEPES) pH 7.2, containing 0.1% porcine gelatin, 0.1% Tween-80, and0.01% sodium azide) and continuing incubation for 1 hour at 37° C. or at4° C. for 1-7 days.

Lyophilized peptides to be tested were weighed in tared 12×75 mmpolystyrene tubes and reconstituted to a concentration of 1.2 mM withassay buffer. Each suspension was sonicated in a water bath for 1-5minutes and vortexed 15-30 seconds to disperse large particles. Serialdilutions of each peptide suspension were prepared using assay buffer ina 96-well polystyrene microtiter plate. Additional wells received Fabfragment of the mouse/human monoclonal anti-human TNFα antibody cA2(positive control) or assay buffer (negative control). Human recombinantTNFα (Biosource, Camarillo, Calif.) was added to all wells to give afinal TNFα concentration of 25 ng/ml, final peptide concentrations of1.0, 0.33 and 0.11 mM and a final cA2 Fab concentration of 500 ng/ml.The polystyrene dilution plate was then sealed and incubated 1 hour atroom temperature on an orbital mixer set at moderate speed.

Following the sample preincubation, blocker was discarded from thep55-Ig coated plate and blotted dry. Aliquots (50 μl) of each peptide orcontrol were transferred into duplicate wells on the p55-Ig coated platewhich was then sealed and incubated 1 hour at 37° C. Duplicate wellscontaining only assay buffer were included as a plate blank. Afterincubation the p55-Ig plate was washed three times with 0.05% Tween-20in PBS.

To detect TNFα captured by the p55-Ig plate, all wells were incubated insuccession with polyclonal rabbit anti-human TNFα (Genzyme, Boston,Mass.; 1:500 in assay buffer, 50 μl/well), biotinylated goat anti-rabbitIg (H&L) (Vector Laboratories, Burlingame, Calif; 1 μg/ml in assaybuffer, 50 μl/well) and streptavidin-alkaline phosphatase conjugate(Pierce, Rockford, Ill.; 1 μg/ml in assay buffer, 50 μl/well). Allincubations were for 1 hour at 37° C. and after each incubation theplate was washed three times with 0.05% Tween-20 in PBS. Fiftymicroliters of alkaline phosphatase substrate solution (alkaline buffer(Sigma, St. Louis, Mo.) diluted 1:500 in deionized water plus one 5 mgtablet of p-nitrophenyl phosphate (Sigma, St. Louis, Mo.) per 5 ml ofdiluted buffer) was added to all wells and incubation continued for 20minutes at room temperature. Color development was stopped by adding toall wells 50 μl of 3N sodium hydroxide. The optical density (OD) at 405nm was measured on a microtiter plate reader (Molecular Devices Vmaxplate reader), with the plate blank wells subtracted as background. Theactivity of each peptide was then expressed as the percent inhibition ofTNFα capture by the p55-Ig plate, relative to the amount of TNFαcaptured in wells containing only assay buffer (negative control), asfollows:

% inhibition=100 - ((mean OD peptide/mean OD negative control)×100).

The validity of each assay was confirmed by the cA2 Fab positive controlwhich typically inhibited TNFα capture by more than≧75%.

Example 2

The inhibition of binding of TNFα to p55TNFr-IgG chimeric construct wasperformed as described above using several embodiments of the invention.The peptides were tested to determine the concentration necessary toinhibit TNFα by 50% (IC₅₀) relative to TNFα activity in the absence ofthe peptide. The following data were generated.

The IC₅₀ of SEQ ID NO:1 in which the carboxy terminal phenylalanine isphenylalanine amide was 0.37 mM.

The IC₅₀ of SEQ ID NO:2 in which the carboxy terminal tyrosine istyrosine amide was 0.85 mM.

The IC₅₀ of SEQ ID NO:3 in which the carboxy terminal alanine is alanineamide was 0.60 mM.

The IC₅₀ of SEQ ID NO:4 in which the carboxy terminal glutamine isglutamine amide was 0.31 mM.

The IC₅₀ of SEQ ID NO:5 in which the carboxy terminal glutamine isglutamine amide was 0.48 mM.

Example 3

The inhibition of binding of TNFα to p55TNFr-IgG chimeric construct wasperformed as described above using various concentrations of severalembodiments of the invention.

The data shown in FIG. 1 demonstrates the dose dependent inhibition ofTNFα by each peptide. The peptides used were:

"C-terminal blocked SEQ ID NO:2" (SEQ ID NO:2 in which the carboxyterminal tyrosine is tyrosine amide);

"C-terminal blocked SEQ ID NO:5" (SEQ ID NO:5 in which the carboxyterminal glutamine is glutamine amide);

Example 4

TNFα cytotoxicity assays

The ability of the compounds of the invention to bind to the TNFreceptor and inhibit activity by human TNFα is tested in a TNFα-mediatedcell killing assay. WEHI-164 murine fibrosarcoma cells (Espevik et al.,J. Immunol. Methods 1986, 95, 99-105), or another cell line sensitive tothe cytotoxic effects of TNF, are used in the following cytotoxicityassay to identify peptides with TNFα antagonist activity. The cells aregrown in Dulbecco's modified Eagle's medium supplemented with 5%heat-inactivated fetal bovine serum, glutamine, nonessential amino acidsand sodium pyruvate (DMEM/FBS). The WEHI cells are harvested using acell scraper and suspend in DMEM/FBS at 1×10⁶ cells/mL. The cells arethen seeded at 50 μL (˜5×10⁴ cells) per well in a 96-well microtiterplace. The plate is then incubated for 3-4 hr at 37° C. in 5% CO₂ oruntil 50% confluent.

The peptides to be tested are solubilized at approximately 2.5 mM in 10mM HEPES pH 7.5 by vortexing and brief sonication. Each peptide is then0.2 μ filtered and the peptide concentration estimated by measuring theabsorbance at 214 nanometers. Two serial threefold dilutions of eachpeptide are prepared in 10 mM HEPES pH 7.5. Four serial twofold.dilutions of an anti-TNFα FAB known to inhibit TNFα activity such as forexample cA2 Fab are also prepared in 10 mM HEPES pH 7.5 to serve as apositive inhibition control. One-fourth volume of DMEM/FBS containing 10μg/mL actinomycin D and 500 pg/mL of recombinant human TNFα is thenmixed with each dilution of peptide and cA2 Fab, as well as with 10 mMHEPES pH 7.5 (TNF control), and preincubated for 30 minutes at roomtemperature. A cell control is also prepared in which one-fourth volumeof DMEM/FBS containing 10 μg/mL of actinomycin D (but no TNF) is addedto 10 mM HEPES pH 7.5.

After preincubation, the peptides and controls are transferred (50μL/well) in triplicate to the microtiter wells seeded with WEHI cellsand incubated overnight at 37° C. in 5% CO₂. Viable cells are detectedusing a 5 mg/mL solution of 3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) in 0.01M sodium phosphate, 0.15M sodiumchloride pH 7.2. After 0.2μ filtration, 25 μL of the MTT solution isadded to each well and incubation continued for 2 hours at 37° C. in 5%CO₂. After incubation, the cells and the blue formazan precipitate aresolubilized by adding 100 μL/well of 20% (w/v) sodium dodecyl sulfatedissolved in 50% (v/v) dimethylformamide in water. The absorbance at 570nm (corrected for scatter at 630 nm) is a direct measure of the numberof cells that survived in each well. Replicates are averaged and thefraction of cells that survive is calculated based on the absorbanceobtained in the cell control wells. The percent inhibition of TNFαactivity is then calculated as:

    ______________________________________                                        fraction of surviving cells                                                                     ×                                                                              [1.0-fraction of                                     in presence of peptide   cells in TNF control].                               surviving                                                                     ______________________________________                                    

Example 5

Treatment of Arthritis, Sepsis, Allograft Rejection and Graft VersusHost Disease

In rheumatoid arthritis, the main presenting symptoms are pain,stiffness, swelling, and loss of function (Bennett JC. The etiology ofrheumatoid arthritis, in Textbook of Rheumatology, Kelley WN, Harris ED,Ruddy S, Sledge CB, Eds., WB Saunders, Philadelphia, 1985, pp 879-886).The multitude of drugs used in controlling such symptoms seems largelyto reflect the fact that none is ideal. Although there have been manyyears of intense research into the biochemical, genetic,microbiological, and immunological aspects of rheumatoid arthritis, itspathogenesis is not completely understood, and none of the treatmentsclearly stop progression of joint destruction (Harris ED, RheumatoidArthritis: The clinical spectrum, in Textbook of Rheumatology, KelleyWN, Harris ED, Ruddy S, Sledge CB, Eds., WB Saunders, Philadelphia,1985, pp 915-990) .

TNFα is of major importance in the pathogenesis of rheumatoid arthritis.TNFα is present in rheumatoid arthritis joint tissues and synovial fluidat the protein and mRNA level (Buchan G, Barrett K, Turner M, Chantry D,Naini RN, and Feldmann N., Interleukin-1 and tumor necrosis factor mRNAexpression in rheumatoid arthritis: prolonged production of IL-1α, Clin.Exp. Immunol. 1988, 73, 449-455), indicating local synthesis. However,detecting TNFα in rheumatoid arthritis joints even in quantitiessufficient for bioactivation does not necessarily indicate that it isimportant in the pathogenesis of rheumatoid arthritis, nor that it is agood candidate therapeutic target. In order to address these questions,the effects of anti-TNFα antibody (rabbit or monoclonal) on rheumatoidjoint cell cultures, and for comparison, osteoarthritic cell cultures,have been studied. The initial result, that IL-I production wasabolished, suggested that TNFα was a therapeutic target for the therapyof rheumatoid arthritis, since anti-TNFα would block both TNFα and IL-I,the two cytokines known to be involved in cartilage and bone destruction(Brennan FM, Chantry D, Jackson A, Maini RN, and Feldmann M, Inhibitoryeffect of TNFα antibodies on synovial cell interleukin-1 production inrheumatoid arthritis, Lancet 1989, II, 244-247).

Subsequent studies in rheumatoid arthritis tissues have supported thishypothesis. Thus it was found that anti-TNFα abrogated the production ofanother proinflammatory cytokine, GM-CSF (Haworth C, Brennan FM, ChantryD, Maini RN, and Feldmann M, GM-CSF expression in rheumatoid arthritis:regulation by tumor necrosis factor alpha, Eur. J. Immunol. 1991, 21,2575-2579). This observation has been independently confirmed(Alvaro-Gracia et al., Cytokines in chronic inflammatory arthritis, VI.1991, Analysis of synovial cell involved in granulocyte-macrophagecolony-stimulating factor production and gene expression in rheumatoidarthritis and its regulation by IL-I and tumor necrosis factor-α). Ithas also been demonstrated that anti-TNFα diminishes cell adhesion andHLA class II expression in rheumatoid arthritis joint cell cultures.

Example 6

Treatment of HIV Infection

TNFα is capable of inducing HIV expression in HIV-infected cell lines.See Poli et al., Proc. Natl. Acad. Sci. USA 1990 87, 782-785 and Buteraet al., J. Immunology 1993 150, 625-634. Butera et al. demonstrated areduction of induced TNFα production and HIV expression in an infectedcell line after treatment with soluble TNF receptors. Thus, themolecules of the present invention may be used to decrease theexpression of TNFα and thereby lessen the induction of HIV expression.

Example 7

Diagnostic Methods

The present invention also provides the peptide-based inhibitors of TNFαincluding fragments, derivatives, and mimetics thereof which aredetectably labeled, as described below, for use in diagnostic methodsfor detecting TNFα in patients known to be or suspected of having a TNFα-mediated condition.

The detectably labelled molecules of the present invention are usefulfor immunoassays which detect or quantitate TNFα in a sample. Animmunoassay for TNFα typically comprises incubating a biological samplein the presence of a detectably labeled high affinity molecule of thepresent invention capable of selectively binding to TNF, and detectingthe labeled molecules which is bound in a sample. Various clinicalimmunoassay procedures are described in Immunoassays for the 80's AVoller et al., Eds., University Park, 1981.

Thus, in this aspect of the invention, the molecule or a biologicalsample may be added to nitrocellulose, or other solid support which iscapable of immobilizing cells, cell particles or soluble proteins. Thesupport may then be washed with suitable buffers followed by treatmentwith the detectably labeled TNFα-specific antibody. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on said solid support maythen be detected by conventional means.

By "solid phase support" or "carrier" is intended any support capable ofbinding TNFα proteins or molecules of the present invention. Well-knownsupports or carriers, include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to TNFα or an anti-TNFα antibody. Thus, the supportconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports include polystyrene beads. Those skilled in the artwill know many other suitable carriers for binding TNFα or compounds ofthe invention, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of anti-TNFα compound may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

One of the ways in which the TNFα-specific molecule can be detectablylabeled is by linking the same to an enzyme and use in an enzymeimmunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA). Thisenzyme, when subsequently exposed to its substrate, will react with thesubstrate generating a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used to detectably label the TNFα-specific molecules of thepresent invention include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. By radioactively labeling the TNFα-specificmolecules, it is possible to detect TNFα through the use of aradioimmunoassay (RIA) (see, for example, Work, T. S., et al.,Laboratory Techniques and Biochemistry in Molecular Biology, NorthHolland Publishing Company, N.Y., 1978. The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography. Isotopes which are particularly usefulfor the purpose of the present invention are: ³ H, ¹²⁵ I, ¹³¹ I, ³⁵ S,¹⁴ C, and, preferably, ¹²⁵ I.

It is also possible to label the TNFα-specific molecules with afluorescent compound. When the fluorescent labeled compound is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labellingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The TNFα-specific molecules can also be detectably labeled usingfluorescence-emitting metals such as ¹⁵² Eu, or others of the lanthanideseries. These metals can be attached to the TNFα-specific molecule usingsuch metal chelating groups as diethylenetriaminepentaacetic acid (DTPA)or ethylenediamine-tetraacetic acid (EDTA).

The TNFα-specific molecules also can be detectably labeled by couplingto a chemiluminescent compound. The presence of the chemiluminescentlylabeled compound is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

Likewise, a bioluminescent compound may be used to label theTNFα-specific molecule, fragment or derivative of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin. Detection of the TNFα-specific compound,fragment or derivative may be accomplished by a scintillation counter,for example, if the detectable label is a radioactive gamma emitter, orby a fluorometer, for example, if the label is a fluorescent material.In the case of an enzyme label, the detection can be accomplished bycolorometric methods which employ a substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

For the purposes of the present invention, the TNFα which is detected bythe above assays may be present in a biological sample. Any samplecontaining TNFα can be used. Preferably, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, inflammatoryexudate, cerebrospinal fluid, amniotic fluid, a tissue extract orhomogenate, and the like. However, the invention is not limited toassays using only these samples, it being possible for one of ordinaryskill in the art to determine suitable conditions which allow the use ofother samples.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledantibodies of the present invention to such a specimen. The peptide ispreferably provided by applying or by overlaying the labeled molecule(or fragment) to a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of TNFα butalso the distribution of TNFα in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

The peptide, fragment or derivative of the present invention may beadapted for utilization in an immunometric assay, also known as a"two-site" or "sandwich" assay. In a typical immunometric assay, aquantity of unlabeled peptide (or fragment of the peptide) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantitation of the ternary complex formed between solid-phasepeptide, TNFα, and labeled anti-TNFα antibody.

Typical and preferred immunometric assays include "forward" assays inwhich the peptide of the invention bound to the solid phase is firstcontacted with the sample being tested to extract the TNFα from thesample by formation of a binary solid phase peptide-TNFα complex. Aftera suitable incubation period, the solid support is washed to remove theresidue of the fluid sample, including unreacted TNFα, if any, and thencontacted with the solution containing a known quantity of labeledpeptide (which functions as a "reporter molecule"). After a secondincubation period to permit the labeled peptide to complex with the TNFαbound to the solid support through the unlabeled peptide, the solidsupport is washed a second time to remove the unreacted labeled peptide.This type of forward sandwich assay may be a simple "yes/no" assay todetermine whether TNFα is present or may be made quantitative bycomparing the measure of labeled peptide with that obtained for astandard sample containing known quantities of TNFα. Such "two-site" or"sandwich" assays are described by Wide, Radioimmune Assay Method,Kirkham, Ed., E. & S. Livingstone, Edinburgh, 1970, pp. 199-206).

Other type of "sandwich" assays, which may also be useful with TNFα, arethe so-called' "simultaneous" and "reverse" assays. A simultaneous assayinvolves a single incubation step wherein the peptide bound to the solidsupport and labeled peptide are both added to the sample being tested atthe same time. After the incubation is completed, the solid support iswashed to remove the residue of fluid sample and uncomplexed labeledpeptide. The presence of labeled peptide associated with the solidsupport is then determined as it would be in a conventional "forward"sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeledpeptide to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period, isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled peptide. The determinationof labeled peptide associated with a solid support is then determined asin the "simultaneous" and "forward" assays. In one embodiment, acombination of peptide of the present invention specific for separateepitopes may be used to construct a sensitive three-siteimmunoradiometric assay.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrLeuAlaHis GluValGlnLeuPheSerSerGlnTyrProPhe                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ii ) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValTyrProGlyLeuGlnGluProTrpLeuHisSerMetTyr                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ProGlyLeuGlnGluProTrpLeuHisSerMetTyrHisGlyAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnGluProTrpLeuHisSerMetTyrHisGlyAlaAlaPheGln                                 15 1015                                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TrpLeuHisSerMetTyrHisGlyAlaAlaPheGlnLeuThr Gln                                151015                                                                    

I claim:
 1. A peptide consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:5.
 2. The peptide of claim 1 wherein said peptide is alinear peptide.
 3. The peptide of claim 1 wherein said peptide comprisesa blocked amino terminal residue and/or a blocked carboxy terminalresidue.
 4. The peptide of claim 1 wherein said peptide comprises anacetylated amino terminal residue and an amidated carboxy terminalresidue.
 5. The peptide of claim 1 wherein said peptide is selected fromthe group consisting of: SEQ ID NO:2 in which the carboxy terminaltyrosine is tyrosine amide; SEQ ID NO:3 in which the carboxy terminalalanine is alanine amide SEQ ID NO:4 in which the carboxy terminalglutamine is glutamine amide; and SEQ ID NO:5 in which the carboxyterminal glutamine is glutamine amide.
 6. The peptide of claim 1consisting of SEQ ID NO:2.
 7. The peptide of claim 1 consisting of SEQID NO:3.
 8. The peptide of claim 1 consisting of SEQ ID NO:4.
 9. Thepeptide of claim 1 consisting of SEQ ID NO:5.